The genome of HIV-1 is composed of RNA, and after this RNA enters a cell, it must be converted to viral DNA, which can then insert into the cell's DNA, and be used to produce new viral protein and viruses. The RNA is converted into DNA by the viral enzyme reverse transcriptase. This enzyme requires a primer to start reverse transcription. The primer for reverse transcription in HIV-1 is human tRNALys3, which is selectively packaged into the virion during viral assembly in the cell, and binds (anneals) to a region in the viral RNA genome termed the primer binding site (PBS). It is from this bound tRNALys3 that reverse transcription begins. The selective packaging of tRNALys3 occurs because the viral protein Gag specifically interacts with a major tRNALys3-binding protein in the cell, lysyl-tRNA synthetase (LysRS), so that both tRNALys3 and LysRS are packaged into the assembling virus. Disrupting this reaction through mutations in either Gag or LysRS prevent tRNALys3 from being packaged into the virus, and greatly reduces reverse transcription and viral infectivity. The Gag/LysRS interaction, therefore, represents a novel target for anti-HIV-1 therapy. In this project, we are seeking to identify small molecule inhibitors of this interaction. This will be done using high throughput screening (HTS) of libraries of compounds to find molecules that inhibit the Gag/LysRS interaction. The interaction of LysRS with either Gag or the smaller capsid (CA) molecule will be measured using fluorescent anisotropy (FA), using fluorescently-labeled CA as the tracer in the HTS of the LysRS/CA interaction. The ability of small peptides or RNA molecules to inhibit this interaction will also be investigated, using peptides from Gag or LysRS that are known to be involved in the Gag/LysRS interaction, or SELEX to isolate RNA molecules that specifically bind to CA. Project Narrative Many reports have now demonstrated that the selective packaging into HIV-1 of the primer for reverse transcription, tRNALys3, is essential for the viral life cycle. This selective incorporation of tRNALys3 requires a specific interaction between Gag and LysRS, making the Gag/LysRS interaction an attractive target for antiviral drug development. The feasibility of using a fluorescence anisotropy assay to detect the binding interaction has already been demonstrated, and will be used in this project facilitate the development of rapid screening techniques to search for inhibitors of this novel target, which will then be tested for their ability to specifically inhibit HIV-1 replication.